This application claims priority to U.S. Provisional application No. 60/384,333, filed May 30, 2002.
This invention was made with U.S. Government support under Contract No. DE-FC-36-00GO10598, awarded by the Department of Energy. The U.S. government has certain rights in this invention.
Lactic acid has wide industrial applicability including uses in chemical processing and synthesis, cosmetics, pharmaceuticals, plastics, and food production. Most industrial scale processes for making lactic acid are fermentation processes. Various lactic acid-producing bacteria have been used in those fermentation processes.
Recent research has investigated the use of recombinant yeast strains in lactic acid fermentation processes. Recombinant yeast potentially can provide several advantages over bacterial fermentations. Some yeast strains are more resistant to higher temperatures. This potentially allows for higher temperature fermentations, which can translate to faster rates of fermentations. Better resistance to high temperature can make it easier to purge a fermentation medium of contaminating microbes, as the medium can simply be heated to a temperature at which the unwanted species die off but the desired species can tolerate. Lactic acid-producing bacteria such as lactobacilli require a complex fermentation medium in order to produce efficiently. The complexity of the fermentation medium increases raw materials costs and makes it more difficult and expensive to separate the lactic acid from the medium. Using recombinant yeast offers the possibility of reducing costs by using a simplified fermentation medium.
Porro and coworkers have attempted to engineer a lactic-acid producing yeast by inserting an exogenous LDH (lactate dehydrogenase) gene into yeast cells from the species S. cerevisiae, K. lactic, T. delbrueckii and Z. bailii, and disrupting the cell's natural pyruvate pathway. See Porro et al., “Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid”, Biotechnol. Prog. 1995 May–June; 11(3): 294–8; Porro et al., “Replacement of a metabolic pathway for large-scale production of lactic acid from engineered yeasts”, App. Environ. Microbiol. 1999 Sep:65(9):4211–5; Bianchi et al., “Efficient homolactic fermentation by Kluyveromyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene”, App. Environ. Microbiol. 2001 Dec; 67(12)5621–5. Porro was able to produce a recombinant yeast that produces lactic acid, but the strains did not perform nearly well enough for implementation in any commercial process. To qualify for use in an industrial environment, the strain must generate good yields of lactic acid (i.e., high conversion of the substrate to lactic acid) and high productivity (i.e., rapid metabolism of the substrate to lactic acid). The yeast preferably is able to tolerate a medium having a high titer of lactic acid.
More recently, Rajgarhia and coworkers have created recombinant yeast that exhibits higher yields and productivities than those of Porro. See, for example, WO 00/71738, WO 02/42471 and PCT/US02/16223. Rajgarhia's work as described in WO 00/71738 attempts to take advantage of the so-called “Crabtree negative” phenotype exhibited by certain species of yeast. The Crabtree effect is defined as the occurrence of fermentative metabolism under aerobic conditions due to the inhibition of oxygen consumption by a microorganism when cultured at high specific growth rates (long-term effect) or in the presence of high concentrations of glucose (short-term effect). Crabtree negative phenotypes do not exhibit this effect, and are thus able to consume oxygen even in the presence of high concentrations of glucose or at high growth rates. Thus, cultures of Crabtree negative microorganisms, in theory at least, can be converted from a growth phase to a fermentation (production) phase through manipulation of oxygen supply. In the presence of significant aeration, the microorganisms grow to produce biomass and CO2, whereas under anaerobic conditions, the cells instead ferment the available substrate to produce lactic acid or other fermentation products.
We have found, however, that certain strains do not ferment as efficiently as desired under strictly anaerobic conditions. This is true, for example, in engineered yeast strains in which the pyruvate decarboxylase (PDC) pathway is deleted or disrupted. However, the use of such engineered species is otherwise highly desirable in lactic acid fermentations (as well as others in which the desired product is not ethanol), as disruption of the PDC pathway reduces the amount of ethanol that is produced. Accordingly, it would be desirable to provide an improved fermentation process in which a strain that does not ferment efficiently under strictly anaerobic conditions can produce a desired fermentation product economically.